Inhibitors Of The Influenza A Virus M2 Proton Channel

ABSTRACT

Provided are compounds that are capable of modulating the activity of the influenza A virus via interaction with the M2 transmembrane protein. Also provided are methods for treating an influenza A-affected disease state or infection comprising administering a composition comprising one or more compounds that have been identified as being capable of interaction with the M2 protein.

TECHNICAL FIELD

The present invention pertains to, among other things, compounds andmethods for modulating the activity of the influenza virus.

BACKGROUND

The M2 protein is found in the viral envelope of influenza A virus andfunctions as a highly selective, pH-regulated proton channel importantfor the life cycle of the virus. Unlike neuraminidase inhibitors,rimantadine and amantadine are anti-viral agents capable of blocking thetetrameric M2 channel. In 2006, the CDC issued an alert instructingclinicians to avoid using M2 ion-channel inhibitors during influenzaseason due to the extraordinarily high frequency of amantadineresistance in influenza A isolates associated with a single pointmutation in the M2 protein, S31N (Hayden F. G., Antiviral Resistance inInfluenza Viruses—Implications for Management and Pandemic Response, NEnj J Med, 2006, 354; 8). The drug-binding site is lined by residuesthat are mutated in amantadine-resistant viruses. Grambas, S., Bennett,M. S. & Hay, A. J. Influence of amantadine resistance mutations on thepH regulatory function of the M2 protein of influenza A viruses.Virology 191, 541-549 (1992); Bright, R. A., Shay, D. K., Shu, B., Cox,N. J. & Klimov, A. I. Adamantane resistance among influenza A virusesisolated early during the 2005-2006 influenza season in the UnitedStates. J. Am. Med. Assoc. 295, 891-894 (2006). Recently, it has beenreported that resistance to rimantadine and amantadine in humans, birdsand pigs has reached more than 90%, casting into doubt the continuedability of these drugs alone to satisfy the need for treatment ofinfluenza (Deyde, V. M. et al. Surveillance of resistance to adamantanesamong influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J.Infect. Dis. 196, 249-257 (2007)).

Previous studies have suggested that BL-1743(3-(4,5-Dihydro-1H-imidazol-2-yl)-3-aza-spiro[5.5]undecane) interactsdifferently with the M2 proton channel as compared with amantadine, buthave found that the majority of isolated influenza viruses that areamantadine-resistant are also resistant to BL-1743. Tu Q, et al.,Characterization of inhibition of M2 ion channel activity by BL-1743, aninhibitor of influenza A virus, J. Virol. 1996 July; 70(7):4246-52. Forexample, Tu Q, et al. found that mutations known to confer amantadineresistance at M2 residues 27, 30, 31, and 34, all within the M2transmembrane domain, also induce “complete” resistance to BL-1743. Id.The publication by Tu Q, et al. concluded that “the overlapping spectraof amantadine and BL-1743 resistance mutations and the higher apparentK_(i) . . . do not indicate that BL-1743 should replace the use ofamantadine (or rimantadine) for the prophylaxis or treatment ofinfluenza virus infections in humans.” Id. See also Kurtz, et al.,Growth impairment resulting from expression of influenza virus M2protein in Saccharomyces cerevisiae: identification of a novel inhibitorof influenza virus. Antimicrob Agents Chemother. 1995 October;39(10):2204-9 (“BL-1743 does not produce an additive effect on M2inhibition, suggesting that these two compounds interact with similarsites in the M2 protein . . . . Thus, BL-1743 appears to represent anovel structure with an antiviral profile similar to that ofamantadine.”).

SUMMARY

In one aspect of the present invention, provided are compounds havingthe formula (I):

wherein

X and Y are independently a bond, alkylene, or amine;

R¹ and R² are independently hydrogen, alkyl, hydroxyl, carbonyl,carboxyl, cyano, amino, or —CH(R⁵)(R⁶);

R³ and R⁴ are independently hydrogen or —CH(R⁷)(R⁸);

R⁵ and R⁶ are independently hydrogen, alkyl, hydroxyl, carbonyl, oramino;

R⁷ and R⁸ are independently hydrogen, alkyl, alkoxy, hydroxyl, carbonyl,or amino;

dashed lines a and b are optional bonds; and,

n is 0-3,

or a stereoisomer, partial stereoisomer, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.

In other aspects, methods are provided for treating an influenza Avirus-affected disease state or infection comprising the step ofadministering to a subject in need thereof a composition comprising acompound of formula (I) as described above.

Also disclosed are compositions comprising a compound of formula (I) anda pharmaceutically acceptable carrier, excipient, or diluent.

FIGURES

FIG. 1 depicts the results of a plaque reduction assay of certaincompounds according to the present disclosure on A/M2-V27A/L38F mutantinfluenza virus.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingexamples, which form a part of this disclosure. It is to be understoodthat this invention is not limited to the specific products, methods,conditions or parameters described and/or shown herein, and that theterminology used herein is for the purpose of describing particularembodiments by way of example only and is not intended to be limiting ofthe claimed invention.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in their entirety.

As employed above and throughout the disclosure, the following terms andabbreviations, unless otherwise indicated, shall be understood to havethe following meanings.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “acompound” is a reference to one or more of such compounds andequivalents thereof known to those skilled in the art, and so forth.Furthermore, when indicating that a certain chemical moiety “may be” X,Y, or Z, it is not intended by such usage to exclude other choices forthe moiety; for example, a statement to the effect that R₁ “may bealkyl, aryl, or amino” does not exclude other choices for R₁, such ashalo, aralkyl, and the like.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. As used herein, “about X” (where X is a numerical value)preferably refers to ±10% of the recited value, inclusive. For example,the phrase “about 8” refers to a value of 7.2 to 8.8, inclusive; asanother example, the phrase “about 8%” refers to a value of 7.2% to8.8%, inclusive. Where present, all ranges are inclusive and combinable.For example, when a range of “1 to 5” is recited, the recited rangeshould be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2& 4-5”, “1-3 & 5”, and the like. In addition, when a list ofalternatives is positively provided, such listing can be interpreted tomean that any of the alternatives may be excluded, e.g., by a negativelimitation in the claims. For example, when a range of “1 to 5” isrecited, the recited range may be construed as including situationswhereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, arecitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, orsimply “wherein 2 is not included.” In another example, when a listingof possible substituents including “hydrogen, alkyl, and aryl” isprovided, the recited listing may be construed as including situationswhereby any of “hydrogen, alkyl, and aryl” is negatively excluded; thus,a recitation of “hydrogen, alkyl, and aryl” may be construed as“hydrogen and aryl, but not alkyl”, or simply “wherein the substituentis not alkyl”.

Protective groups are abbreviated according to the system disclosed inGreene, T. W. and Wuts, P. G. M, Protective Groups in Organic Synthesis2d. Ed., Wiley & Sons, 1991, which is incorporated in its entiretyherein. For example, “CBZ” or “Cbz” or “Z” stands for carbobenzyloxy orbenzyloxycarbonyl, “Boc” or “BOC” represents t-butoxycarbonyl, “Alloc”denotes allyloxycarbonyl, Bz means benzoyl, and “Fmoc” stands for9-fluorenylmethoxycarbonyl.

As used herein, the terms “component,” “composition of compounds,”“compound,” “drug,” “pharmacologically active agent,” “active agent,”“therapeutic,” “therapy,” “treatment,” or “medicament” are usedinterchangeably herein to refer to a compound or compounds orcomposition of matter which, when administered to a subject (human oranimal) induces a desired pharmacological and/or physiologic effect bylocal and/or systemic action.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “min” means minute(s),“g” means gram(s), “mg” means milligram(s), “μg” means microgram(s),“eq” means equivalent(s), “h” means hour(s), “μL” means microliter(s),“mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmol”or “mmole” means millimole(s), “cm” means centimeters, “SEM” meansstandard error of the mean, and “IU” means International Units. “IC₅₀value” or “IC₅₀” means dose of the compound which results in 50%alleviation or inhibition of the observed condition or effect.

As used herein, “alkyl” refers to an optionally substituted, saturatedstraight, or branched, hydrocarbon radical having from about 1 to about20 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein). Where appropriate, “alkyl”can mean “alkylene”; for example, if X is —R₁R₂, and R₁ is said to be“alkyl”, then “alkyl” may correctly be interpreted to mean “alkylene”.

“Amino” refers to —NH₂ and may include one or more substituents thatreplace hydrogen. “Amino” may be used interchangeably with “amine” andis also intended to include any pharmaceutically acceptable amine salts.For example, amino/amine may refer to —NH⁺(X)(Y)Cl⁻, —NH—, or—NH⁺(X)Cl⁻—, wherein X and Y are preferably and independently hydrogenor alkyl, wherein alkyl may include one or more halo substitutions.

As used herein, “aryl”, “arene”, and “aromatic” each refer to anoptionally substituted, saturated or unsaturated, monocyclic,polycyclic, or other homo- or heterocyclic aromatic ring system havingfrom about 3 to about 50 ring members (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from about 5 to about 10 ring atom members being preferred. Suchmoieties encompass (include) “heteroaryl” and “heteroarene” as definedinfra. Where appropriate, “aryl” can mean “arene”; for example, if X is—R₁R₂, and R₁ is said to be “aryl”, then “aryl” may correctly beinterpreted to mean “arene”.

As used herein, “alkenyl” refers to an alkyl radical having from about 2to about 20 carbon atoms and one or more double bonds (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), wherein alkyl is as previously defined. In someembodiments, it is preferred that the alkenyl groups have from about 2to about 6 carbon atoms. Alkenyl groups may be optionally substituted.

As used herein, “aralkyl” refers to alkyl radicals bearing one or morearyl substituents and having from about 4 to about 50 carbon atoms (andall combinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), wherein aryl and alkyl are as previously defined.In some preferred embodiments, the alkyl moieties of the aralkyl groupshave from about 1 to about 4 carbon atoms. In other preferredembodiments, the alkyl moieties have from about 1 to about 3 carbonatoms. Aralkyl groups may be optionally substituted.

“Alkylamino” signifies alkyl-(NH)—, wherein alkyl is as previouslydescribed and NH is defined in accordance with the provided definitionof amino. “Arylamino” represents aryl-(NH)—, wherein aryl is as definedherein and NH is defined in accordance with the provided definition ofamino. Likewise, “aralkylamino” is used to denote aralkyl-(NH)—, whereinaralkyl is as previously defined and NH is defined in accordance withthe provided definition of amino. “Alkylamido” refers toalkyl-CH(═O)NH—, wherein alkyl is as previously described. “Alkoxy” asused herein refers to the group R—O— where R is an alkyl group, andalkyl is as previously described. “Aralkoxy” stands for R—O—, wherein Ris an aralkyl group as previously defined. “Alkylsulfonyl” meansalkyl-SO₂—, wherein alkyl is as previously defined.

As used herein, “alkylene” refers to an optionally branched orsubstituted bivalent alkyl radical having the general formula—(CH₂)_(n)—, where n is 1 to 10. Non-limiting examples includemethylene, trimethylene, pentamethylene, and hexamethylene.

As used herein, “heteroaryl” or “heteroarene” refers to an aryl radicalwherein in at least one of the rings, one or more of the carbon atomring members is independently replaced by a heteroatom group selectedfrom the group consisting of S, O, N, and NH, wherein aryl is aspreviously defined. Heteroaryl/heteroarene groups having a total of fromabout 3 to about 14 carbon atom ring members and heteroatom ring membersare preferred. Likewise, a “heterocyclic ring” is an aryl radicalwherein one or more of the carbon atom ring members may be (but are notnecessarily) independently replaced by a heteroatom group selected fromthe group consisting of S, O, N, and NH. Heterocyclic rings having atotal from about 3 to 14 ring members and heteroatom ring members arepreferred, but not necessarily present; for example, “heterocyclohexyl”may be a six-membered aryl radical with or without a heteroatom group.

“Halo” and “halogen” each refers to a fluoro, chloro, bromo, or iodomoiety, with fluoro, chloro, or bromo being preferred.

“Haloalkyl” signifies halo-alkyl- wherein alkyl and halo, respectively,are as previously described.

The phrase reading “[moiety] is absent” means that the substituents towhich the moiety is attached may be directly attached to each other.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo (e.g., F, Cl, Br, I), alkyl, cycloalkyl, alkylcycloalkyl,cycloalkylalkyl, alkenyl, alkynyl, aralkyl, aryl, heteroaryl,heteroaralkyl, spiroalkyl, heterocycloalkyl, hydroxyl (—OH), nitro(—NO₂), cyano (—CN), amino (—NH₂), —N-substituted amino (—NHR″),—N,N-disubstituted amino (—N(R″)R″), oxo (═O), carboxy (—COOH),—O—C(═O)R″, —C(═O)R″, —OR″, —C(═O)OR″, -(alkylene)-C(═O)—OR″,—NHC(═O)R″, aminocarbonyl (—C(═O)NH₂), —N-substituted aminocarbonyl(—C(═O)NHR″), —N,N-disubstituted aminocarbonyl (—C(═O)N(R″)R″), thiol,thiolato (—SR″), sulfonic acid (—SO₃H), phosphonic acid (—PO₃H),—P(═O)(OR″)OR″, —S(═O)R″, —S(═O)₂R″, —S(═O)₂NH₂, —S(═O)₂ NHR″,—S(═O)₂NR″R″, —NHS(═O)₂R″, —NR″S(═O)₂R″, —CF₃, —CF₂CF₃, —NHC(═O)NHR″,—NHC(═O)NR″R″, —NR″C(═O)NHR″, —NR″C(═O)NR″R″, —NR″C(═O)R″ and the like.In relation to the aforementioned substituents, each moiety R″ can be,independently, any of H, alkyl, cycloalkyl, alkenyl, aryl, aralkyl,heteroaryl, or heterocycloalkyl, for example.

As used herein, the terms “treatment” or “therapy” (as well as differentword forms thereof) includes preventative (e.g., prophylactic), curativeor palliative treatment.

As employed above and throughout the disclosure the term “effectiveamount” refers to an amount effective, at dosages, and for periods oftime necessary, to achieve the desired result with respect to thetreatment of the relevant disorder, condition, or side effect. It willbe appreciated that the effective amount of components of the presentinvention will vary from patient to patient not only with the particularcompound, component or composition selected, the route ofadministration, and the ability of the components to elicit a desiredresponse in the individual, but also with factors such as the diseasestate or severity of the condition to be alleviated, hormone levels,age, sex, weight of the individual, the state of being of the patient,and the severity of the pathological condition being treated, concurrentmedication or special diets then being followed by the particularpatient, and other factors which those skilled in the art willrecognize, with the appropriate dosage ultimately being at thediscretion of the attendant physician. Dosage regimens may be adjustedto provide the improved therapeutic response. An effective amount isalso one in which any toxic or detrimental effects of the components areoutweighed by the therapeutically beneficial effects. As an example, thecompounds useful in the methods of the present invention areadministered at a dosage and for a time such that the level ofactivation and adhesion activity of platelets is reduced as compared tothe level of activity before the start of treatment.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

Within the present invention, the disclosed compounds may be prepared inthe form of pharmaceutically acceptable salts. “Pharmaceuticallyacceptable salts” refer to derivatives of the disclosed compoundswherein the parent compound is modified by making acid or base saltsthereof. Examples of pharmaceutically acceptable salts include, but arenot limited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, such conventional non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Thesephysiologically acceptable salts are prepared by methods known in theart, e.g., by dissolving the free amine bases with an excess of the acidin aqueous alcohol, or neutralizing a free carboxylic acid with analkali metal base such as a hydroxide, or with an amine.

Compounds described herein throughout, can be used or prepared inalternate forms. For example, many amino-containing compounds can beused or prepared as an acid addition salt. Often such salts improveisolation and handling properties of the compound. For example,depending on the reagents, reaction conditions and the like, compoundsas described herein can be used or prepared, for example, as theirhydrochloride or tosylate salts. Isomorphic crystalline forms, allchiral and racemic forms, N-oxide, hydrates, solvates, and acid salthydrates, are also contemplated to be within the scope of the presentinvention.

Certain acidic or basic compounds of the present invention may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention. It is well known in the art that compounds containing bothamino and carboxy groups often exist in equilibrium with theirzwitterionic forms. Thus, any of the compounds described hereinthroughout that contain, for example, both amino and carboxy groups,also include reference to their corresponding zwitterions.

“Hydrate” refers to a compound of the present invention which isassociated with water in the molecular form, i.e., in which the H—OHbond is not split, and may be represented, for example, by the formulaR.H₂O, where R is a compound of the invention. A given compound may formmore than one hydrate including, for example, monohydrates (R.H₂O) orpolyhydrates (R.nH₂O wherein n is an integer>1) including, for example,dihydrates (R.2H₂O), trihydrates (R.3H₂O), and the like, orhemihydrates, such as, for example, R.n_(/2)H₂O, R.n_(/3)H₂O,R.n_(/4)H₂O and the like wherein n is an integer.

“Solvate” refers to a compound of the present invention which isassociated with solvent in the molecular form, i.e., in which thesolvent is coordinatively bound, and may be represented, for example, bythe formula R.(solvent), where R is a compound of the invention. A givencompound may form more than one solvate including, for example,monosolvates (R.(solvent)) or polysolvates (R.n(solvent)) wherein n isan integer>1) including, for example, disolvates (R.2(solvent)),trisolvates (R.3(solvent)), and the like, or hemisolvates, such as, forexample, R.n_(/2)(solvent), R.n_(/3)(solvent), R.n_(/4)(solvent) and thelike wherein n is an integer. Solvents herein include mixed solvents,for example, methanol/water, and as such, the solvates may incorporateone or more solvents within the solvate.

“Acid hydrate” refers to a complex that may be formed throughassociation of a compound having one or more base moieties with at leastone compound having one or more acid moieties or through association ofa compound having one or more acid moieties with at least one compoundhaving one or more base moieties, said complex being further associatedwith water molecules so as to form a hydrate, wherein said hydrate is aspreviously defined and R represents the complex herein described above.

The term “stereoisomers” refers to compounds that have identicalchemical constitution, but differ as regards the arrangement of theatoms or groups in space.

“Racemic” means having the capacity for resolution into forms of opposedoptical activity.

As used herein, the term “partial stereoisomer” refers to stereoisomershaving two or more chiral centers wherein at least one of the chiralcenters has defined stereochemistry (i.e., R or S) and at least one hasundefined stereochemistry (i.e., R or 5). When the term “partialstereoisomers thereof” is used herein, it refers to any compound withinthe described genus whose configuration at chiral centers with definedstereochemistry centers is maintained and the configuration of eachundefined chiral center is independently selected from R or S. Forexample, if a stereoisomer has three chiral centers and thestereochemical configuration of the first center is defined as having“S” stereochemistry, the term “or partial stereoisomer thereof” refersto stereoisomers having SRR, SRS, SSR, or SSS configurations at thethree chiral centers, and mixtures thereof.

“Prodrug” refers to compounds which are themselves inactive or minimallyactive for the activity desired, but through biotransformation can beconverted into biologically active metabolites. For example, a prodrugof the present invention would include, inter alia, any compound whichis convertible in vivo by metabolic means to a compound claimed ordescribed in the present disclosure.

“N-oxide” refers to compounds wherein the basic nitrogen atom of eithera heteroaromatic ring or tertiary amine is oxidized to give a quaternarynitrogen bearing a positive formal charge and an attached oxygen atombearing a negative formal charge.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other occurrence. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

The term “administering” means either directly administering a compoundor composition of the present invention, or administering a prodrug,derivative or analog which will form an equivalent amount of the activecompound or substance within the body.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit maycontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention may be dictated by (a) the unique characteristicsof the active compound(s) and the particular therapeutic effect(s) to beachieved, and (b) the limitations inherent in the art of compoundingsuch active compound(s).

“Subject” or “patient” refers to an embryonic, immature, or adultanimal, including the human species, that is treatable with thecompositions, and/or methods of the present invention.

Accordingly, in one aspect there are provided compounds having theformula

wherein

X and Y are independently a bond, alkylene, or amine;

R¹ and R² are independently hydrogen, alkyl, hydroxyl, carbonyl,carboxyl, cyano, amino, or —CH(R⁵)(R⁶);

R³ and R⁴ are independently hydrogen or —CH(R⁷)(R⁸);

R⁵ and R⁶ are independently hydrogen, alkyl, hydroxyl, carbonyl, oramino;

R⁷ and R⁸ are independently hydrogen, alkyl, alkoxy, hydroxyl, carbonyl,or amino;

dashed lines a and b are optional bonds; and,

n is 0-3,

or a stereoisomer, partial stereoisomer, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.

With respect to the compounds according to formula (I), certain provisosmay apply. These provisos may also optionally apply pursuant to thepresently disclosed methods. For example, if n is 1, both X and Y aremethylene, and both R³ and R⁴ are hydrogen, and one of R¹ and R² ishydrogen, then the other of R¹ and R² is not carbonyl.

In certain embodiments X and Y are each independently methylene orethylene. For example, X is methylene and Y is ethylene, both X and Yare methylene, both X and Y are ethylene, or X is ethylene and Y ismethylene. Where X and Y are each independently methylene or ethylene,for example, at least one of R¹ and R² may be hydrogen. In theseinstances, the other of R¹ and R² may be carbonyl, amino, carboxyl,cyano, or —CH(R⁵)(R⁶). In certain examples wherein at least one of R¹and R² is hydrogen, n is 1 or 2, except that if n is 1, both X and Y aremethylene, and both R³ and R⁴ are hydrogen, then the other of R¹ and R²is not carbonyl. In some instances wherein at least one of R¹ and R² ishydrogen, the other of R¹ and R² is —CH(R⁵)(R⁶). For example, one of R⁵and R⁶ may be amino; in such instances, n may be, for example 1 or 2,and X and Y may independently methylene or ethylene, for example, both Xand Y are methylene. In other instances wherein at least one of R¹ andR² is hydrogen, and the other of R¹ and R² is —CH(R⁵)(R⁶), one of R⁵ andR⁶ may be amino, and the other of R⁵ and R⁶ may be hydrogen.

In other instances wherein X and Y are each independently methylene orethylene, and at least one of R¹ and R² is hydrogen, the other of R¹ andR² is carbonyl; in these examples, if both X and Y are methylene and R³and R⁴ are hydrogen, then n is not 1 or 2. In other examples wherein atleast one of R¹ and R² is hydrogen, and the other of R¹ and R² iscarbonyl, Y may be ethylene.

Where X and Y are each independently methylene or ethylene, and at leastone of R¹ and R² is hydrogen, the other of R¹ and R² may be, forexample, cyano, amino, carboxyl, or —CH(═O)CH₃. In such instances, theother of R¹ and R² may be —NH₃ ⁺Cl⁻.

Exemplary compounds according to the present invention include, amongothers:

wherein n is 0, 2 or 3;

-   -   and stereoisomers, partial stereoisomers, prodrugs,        pharmaceutically acceptable salts, hydrates, solvates, acid        hydrates, and N-oxides thereof.

The compounds employed in the present invention may exist in prodrugform. As used herein, “prodrug” is intended to include any covalentlybonded carriers which release the active parent drug, for example, asaccording to the formulas or compounds employed in the methods of thepresent invention in vivo when such prodrug is administered to asubject. Since prodrugs are known to enhance numerous desirablequalities of pharmaceuticals (e.g., solubility, bioavailability,manufacturing, etc.) the compounds of the present invention may, ifdesired, be delivered in prodrug form. Thus, the present inventioncontemplates methods of delivering prodrugs. Prodrugs of the compoundsemployed in the present invention, for example, according to formula(I), may be prepared by modifying functional groups present in thecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a mammalian subject, cleaves to forma free hydroxyl, free amino, or carboxylic acid, respectively. Examplesinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

As will be readily understood, functional groups present may containprotecting groups during the course of synthesis. Protecting groups areknown per se as chemical functional groups that can be selectivelyappended to and removed from functionalities, such as hydroxyl groupsand carboxyl groups. These groups are present in a chemical compound torender such functionality inert to chemical reaction conditions to whichthe compound is exposed. Any of a variety of protecting groups may beemployed with the present invention. Protecting groups that may beemployed in accordance with the present invention may be described inGreene, T. W. and Wuts, P. G. M, Protective Groups in Organic Synthesis2d. Ed., Wiley & Sons, 1991.

In a further aspect, the invention relates to pharmaceuticalcompositions comprising a compound according to formula (I) and apharmaceutically acceptable carrier, diluent, or excipient. Theapplicable carrier, diluent, or excipient may be selected on the basisof the chosen route of administration and standard pharmaceuticalpractice as described, for example, in Remington's PharmaceuticalSciences (Mack Pub. Co., Easton, Pa., 1985), the disclosure of which ishereby incorporated by reference in its entirety.

Also provided are methods for treating an influenza A virus-affecteddisease state or infection comprising the step of administering to asubject in need thereof a composition comprising a compound of formula(I), wherein any of the embodiments of compounds of formula (I) that aredisclosed herein may be used in accordance with the present methods.

The influenza A virus-affected disease state or infection may compriseany condition that arises as a direct or indirect result of the presenceof influenza A virus. For example, the influenza A virus-affecteddisease state may comprise influenza (flu), pneumonia, bronchitis, sinusinfection, or ear infection, among other conditions. The disease stateor infection may arise as a direct or indirect result of the presence ofwild-type influenza A virus, or may arise as a direct or indirect resultof the presence of a mutant version of the influenza A virus, or mayarise as a direct or indirect result of the presence of both a wild-typeinfluenza A virus and a mutant version of the influenza A virus. Thus,in accordance with the present methods, the influenza A virus may bewild-type or may be a mutant virus. The mutant virus may comprise aninfluenza A virus having the L26F mutation; may comprise an influenza Avirus having the V27G mutation, the V27I mutation, the V27T mutation,the V27S mutation, or the V27A mutation; may comprise an influenza virushaving the A30T mutation; may comprise an influenza virus having theS31A mutation or the S31N mutation; may an influenza virus having theG34E mutation or the G34A mutation; may comprise an influenza virushaving the L38F mutation; may comprise an influenza virus having theW41L mutation or the W41Y mutation; may comprise an influenza virushaving the D44N mutation or the D44H mutation; and/or may comprise aninfluenza virus having the R45K mutation or the R45H mutation.

The compounds of this invention may be administered orally orparenterally, neat or in combination with conventional pharmaceuticalcarriers, diluents, or excipients, which may be liquid or solid. Theapplicable solid carrier, diluent, or excipient may function as, amongother things, a binder, disintegrant, filler, lubricant, glidant,compression aid, processing aid, color, sweetener, preservative,suspensing/dispersing agent, tablet-disintegrating agent, encapsulatingmaterial, film former or coating, flavors, or printing ink. Of course,any material used in preparing any dosage unit form is preferablypharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations. Parenteraladministration in this respect includes administration by, inter alia,the following routes: intravenous, intramuscular, subcutaneous,intraocular, intrasynovial, transepithelial including transdermal,ophthalmic, sublingual and buccal; topically including ophthalmic,dermal, ocular, rectal and nasal inhalation via insufflation, aerosol,and rectal systemic.

In powders, the carrier, diluent, or excipient may be a finely dividedsolid that is in admixture with the finely divided active ingredient. Intablets, the active ingredient is mixed with a carrier, diluent orexcipient having the necessary compression properties in suitableproportions and compacted in the shape and size desired. For oraltherapeutic administration, the active compound may be incorporated withthe carrier, diluent, or excipient and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. The amount of active compound(s) in suchtherapeutically useful compositions is preferably such that a suitabledosage will be obtained. The therapeutic compositions preferably containup to about 99% of the active ingredient.

Liquid carriers, diluents, or excipients may be used in preparingsolutions, suspensions, emulsions, syrups, elixirs, and the like. Theactive ingredient of this invention can be dissolved or suspended in apharmaceutically acceptable liquid such as water, an organic solvent, amixture of both, or pharmaceutically acceptable oils or fat. The liquidcarrier, excipient, or diluent can contain other suitable pharmaceuticaladditives such as solubilizers, emulsifiers, buffers, preservatives,sweeteners, flavoring agents, suspending agents, thickening agents,colors, viscosity regulators, stabilizers, or osmo-regulators.

Suitable solid carriers, diluents, and excipients may include, forexample, calcium phosphate, silicon dioxide, magnesium stearate, talc,sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose,ethylcellulose, sodium carboxymethyl cellulose, microcrystallinecellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins,croscarmellose carbon, acacia, pregelatinized starch, crospovidone,HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminummethahydroxide, agar-agar, tragacanth, or mixtures thereof.

Suitable examples of liquid carriers, diluents and excipients for oraland parenteral administration include water (particularly containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil), or mixturesthereof.

For parenteral administration, the carrier, diluent, or excipient canalso be an oily ester such as ethyl oleate and isopropyl myristate. Alsocontemplated are sterile liquid carriers, diluents, or excipients, whichare used in sterile liquid form compositions for parenteraladministration. Solutions of the active compounds as free bases orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. A dispersioncan also be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations may contain a preservative to prevent the growthof microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form is preferably sterile and fluid toprovide easy syringability. It is preferably stable under the conditionsof manufacture and storage and is preferably preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier, diluent, or excipient may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of adispersion, and by the use of surfactants. The prevention of the actionof microorganisms may be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions may be achieved bythe use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amounts, in the appropriate solvent, withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions may be prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation may include vacuum drying and the freeze dryingtechnique that yields a powder of the active ingredient or ingredients,plus any additional desired ingredient from the previouslysterile-filtered solution thereof.

The compounds of the invention may be administered in an effectiveamount by any of the conventional techniques well-established in themedical field. The compounds employed in the methods of the presentinvention including, for example, the compounds of formula I or II maybe administered by any means that results in the contact of the activeagents with the agents' site or sites of action in the body of apatient. The compounds may be administered by any conventional meansavailable.

Preferably the pharmaceutical composition is in unit dosage form, e.g.as tablets, buccal tablets, troches, capsules, elixirs, powders,solutions, suspensions, emulsions, syrups, wafers, granules,suppositories, or the like. In such form, the composition is sub-dividedin unit dose containing appropriate quantities of the active ingredient;the unit dosage forms can be packaged compositions, for example packetedpowders, vials, ampoules, prefilled syringes or sachets containingliquids. The unit dosage form can be, for example, a capsule or tabletitself, or it can be the appropriate number of any such compositions inpackage form. In addition, dosage forms of the present invention can bein the form of capsules wherein one active ingredient is compressed intoa tablet or in the form of a plurality of microtablets, particles,granules or non-perils. These microtablets, particles, granules ornon-perils are then placed into a capsule or compressed into a capsule,possibly along with a granulation of the another active ingredient.

The dosage of the compounds of the present invention that will be mostsuitable for prophylaxis or treatment will vary with the form ofadministration, the particular compound chosen and the physiologicalcharacteristics of the particular patient under treatment. Generally,small dosages may be used initially and, if necessary, increased bysmall increments until the desired effect under the circumstances isreached. Generally speaking, oral administration may require higherdosages.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations. The dose may also be provided by controlled release ofthe compound, by techniques well known to those in the art.

The compounds useful in the methods of the present invention may beprepared in a number of ways well known to those skilled in the art. Thecompounds can be synthesized, for example, by the methods as describedbelow, or variations thereon as appreciated by the skilled artisan. Thereagents used in the preparation of the compounds of this invention canbe either commercially obtained or can be prepared by standardprocedures described in the literature. All processes disclosed inassociation with the present invention are contemplated to be practicedon any scale, including milligram, gram, multigram, kilogram,multikilogram or commercial industrial scale.

The present invention is further defined in the following Examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only, andshould not be construed as limiting the appended claims From the abovediscussion and these examples, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various usages and conditions.

Examples

All chemicals for use in preparing the inventive compounds werepurchased from commercial vendors and used without further purification,unless otherwise noted.

Example 1 Synthesis and Inhibition Activity of Exemplary Influenza A M2Proton Channel Inhibitors

All chemicals were purchased from commercial vendors and used withoutfurther purification unless otherwise noted. ¹H and ¹³C NMR spectra wererecorded on a DMX-360 NMR spectrometer. Chemical shifts are reported inparts per million referenced with respect to residual solvent (CHCl₃)7.26 ppm and (DMSO-d₆) 2.50 ppm or from internal standardtetramethylsilane (TMS) 0.00 ppm. The following abbreviations were usedin reporting spectra: s) singlet, d) doublet, t) triplet, q) quartet, m)multiplet, d₆) doublet of doublets. All reactions were carried out undera N₂ atmosphere, unless otherwise stated. HPLC grade solvents were usedfor all the reactions. Column chromatography was performed using silicagel (230-400 mesh). Low-resolution mass spectra were obtained using anESI technique on 3200 Q Trap LC/MS/MS system (applied biosystem).

Synthesis of some embodiments was accomplished as illustrated in thefollowing generalized schematic and as described below:

The following schematic represents the process by which some additionalinventive compounds were prepared:

The general procedure for the acid catalyzed robinson annulationreaction is as follows:

Carbaldehyde (10 mmol), but-3-en-2-one (10 mmol) was refluxed in benzene(10 ml) with catalytic amount H₂SO₄ (0.05 ml) in a round bottom flaskequipped with Dean-Stark trap. The mixture was first heated to 45° C.for 1.5 hrs, and then increased to reflux until no more H₂O condensedfrom the reaction mixture (1.5 hr). Another equivalent of bu-3-2-one (10mmol) was added and refluxed for an additional 1.5 hrs. The mixture wascooled down and 20 ml of 1M NaHCO₃ was added and the organic phase wasseparated. The aqueous phase was extracted with benzene, and thecombined organic phase was washed with brine and dried over MgSO₄. Thesolid was filtered, and concentrated in vacuo to give crude dark brownoil which was separated by flash column chromatography (Ethylacetate/Hexane=15% to 25%) to give spiro enone as yellow oil.

Spiro[4.5]dec-6-en-8-one (65% Yield)

¹H-NMR (360 MHz, CDCl₃) δ 6.74 (d, J=7.2 Hz, 2H), 5.84 (d, J=7.2 Hz,2H), 2.44 (t, J=7.2 Hz, 2H), 1.89 (t, J=7.2 Hz, 2H), 1.76-1.67 (m, 8H);¹³C-NMR (90 MHz, CDCl₃) δ 200.19, 159.78, 126.76, 44.32, 38.31, 35.59,34.15, 24.73; ESI-MS: Calculated for C₁₀H₁₄O (M+H)⁺151.2. Found: 151.3

Spiro[5.6]dodec-1-en-3-one

¹H-NMR (360 MHz, CDCl₃) δ 6.75 (d, J=7.2 Hz, 2H), 5.79 (d, J=7.2 Hz,2H), 2.39 (t, J=7.2 Hz, 2H), 1.81 (t, J=7.2 Hz, 2H), 1.62-1.49 (m, 12H);¹³C-NMR (90 MHz, CDCl₃) δ 199.89, 159.40, 126.02, 38.26, 37.81, 34.18,33.61, 30.65, 23.10; ESI-MS: Calculated for C₁₂H₁₈O (M+H)⁺179.3. Found:179.3

The general procedure for reduction of spiro enone with PdCl₂/Et₃SiH isas follows:

To spiro enone (10 mmol) in absolute EtOH was added PdCl₂ (0.2 g),Et₃SiH (20 mmol) was added dropwise to the mixture with stirring. Themixture was refluxed for 4 hrs and PdCl₂ was filtered, washed with EtOH.The filtrate was concentrated and purified by flash columnchromatography (Ethyl acetate/Hexane=15% to 25%) to give spiro ketone asyellow oil.

Spiro[4.5]decan-8-one (85% Yield)

¹H-NMR (360 MHz, CDCl₃) δ 2.34 (t, J=7.2 Hz, 2H), 1.76 (t, J=7.2 Hz,2H), 1.71-1.67 (m, 4H), 1.59-1.55 (m, 4H); ¹³C-NMR (90 MHz, CDCl₃) δ212.97, 42.15, 39.31, 37.95, 37.48, 24.73; ESI-MS: Calculated forC₁₀H₁₆O (M+H)⁺153.2. Found: 153.2.

Spiro[5.6]dodecan-3-one (88% Yield)

¹H-NMR (360 MHz, CDCl₃) δ 2.31 (t, J=7.2 Hz, 2H), 1.67 (t, J=7.2 Hz,2H), 1.57-1.50 (m, 12H); ¹³C-NMR (90 MHz, CDCl₃) δ 213.32, 38.65, 37.81,37.71, 35.25, 30.66, 23.17; ESI-MS: Calculated for C₁₂H₂₀O (M+H)⁺181.3.Found: 181.3

The general procedure for reductive amination of spiro ketone is asfollows:

To a solution of spiro ketone (10 mmol) in EtOH (50 ml) was addedHONH₂*HCl (30 mmol) and NaOAc (40 mmol). The mixture was heated toreflux for 2 hrs. Solvent was removed in vacuo and the residue wasextracted with CH₂Cl₂ and H₂O. The aqueous layer was extracted withCH₂Cl₂ again. The combined CH₂Cl₂ was dried with MgSO₄ and concentratedin vacuo. The resulting oxime was dissolved in anhydrous THF and cooleddown to 0° C. LiAlH₄ (0.80 g, 20 mmol) was added portionwise to thesolution and the mixture was heated to reflux overnight. After coolingdown to 0° C., the solution was quenched with H₂O (0.8 mL), 15% NaOH(0.8 mL), and H₂O (2.4 mL) sequentially. The resulting slurry wasfiltered. The filtrate was acidified with 4M HCl in 1,4-dioxane andconcentrated in vacuo. The final product was purified by flashchromatography to give spirane amine hydrochloride salt as yellow solid.

Spiro[4,5]decan-8-aminium chloride (The Preceding Molecule Wherein n=0)(Yield: 75%)

¹H-NMR (360 MHz, CDCl₃) δ 3.12-3.08 (m, 1H), 1.67-1.45 (m, 16H); ¹³C-NMR(90 MHz, CDCl₃) δ 52.53, 42.96, 37.54, 36.52, 29.96, 26.63; ESI-MS:Calculated for C₁₀H₁₉N (M+H)⁺154.3. Found: 154.3.

Spiro[5.5]undecan-3-aminium chloride

Spiro[5.6]dodecan-3-aminium chloride (Yield: 72%)

¹H-NMR (360 MHz, CDCl₃) δ 3.12-3.08 (m, 1H), 1.67-1.45 (m, 16H); ¹³C-NMR(90 MHz, CDCl₃) δ 52.70, 45.53, 37.93, 36.51, 36.24, 32.64, 32.32,28.20, 24.74, 24.55; ESI-MS: Calculated for C₁₂H₂₃N (M+H)⁺182.3. Found:182.3.

The compound 3-azaspiro[6.6]tridecan-3-ium chloride was prepared asfollows:

To a solution of hydroxylamine-O-sulfonic acid (1.70 g, 15 mmol) and 95%formic acid (9 ml) under N₂ protection was added dropwisespiro[5.6]dodecan-3-one (1.80 g, 10 mmol) in 3 ml of 95% formic acid.The solution was heated under reflux for 5 hrs and then cooled toambient temperature. The reaction mixture was diluted with H₂O (15 ml)and neutralized to pH 7 with 6 N NaOH. The aqueous solution wasextracted three times with 30 ml CHCl₃. The combined organic layer wasdried with MgSO₄ and used for the next step reduction withoutpurification. 3-azaspiro[6.6]tridecan-4-one was dissolved in anhydrousTHF and cooled down to 0° C. LiAlH₄ (0.80 g, 20 mmol) was addedportionwise to the solution and the mixture was heated to refluxovernight. After cooling down to 0° C., the solution was quenched withH₂O (0.8 mL), 15% NaOH (0.8 mL), and H₂O (2.4 mL) sequentially. Theresulting slurry was filtered. The filtrate was acidified with 4M HCl in1,4-dioxane and concentrated in vacuo. The final product was purified byflash chromatography to give 3-azaspiro[6.6]tridecan-3-ium chloride asyellow solid (1.35 g, 62% Yield). ¹H-NMR (360 MHz, CD₃OD) δ 3.31-3.27(m, 2H), 3.12-3.10 (m, 2H), 1.73-1.65 (m, 4H), 1.52-1.47 (m, 6H),1.30-1.27 (m, 8H); ¹³C-NMR (90 MHz, CD₃OD) δ 43.42, 40.75, 39.52, 39.47,37.51, 36.64, 32.18, 23.76, 21.73; ESI-MS: Calculated for C₁₂H₂₃N(M+H)⁺182.3. Found: 182.5.

The general procedure for the reaction used to accomplish ring expansionwas as follows:

To a solution of spiro ketone (5 mmol) and ethyl diazoacetate (1.15 g,10 mmol) in anhydrous THF (20 ml) at −78° C. was added LDA (5 ml, 2M inhexane) dropwise under N₂ atmosphere. The reaction was stirred at thesame temperature for 2 hrs and warmed to ambient temperature. AqueousNH₄Cl (40 ml, 30% w/w) was added and extracted with diethyl ether. Thecombined organic layers were washed with water, brine, dried overanhydrous MgSO₄, filtered and concentrated in vacuo to give thediazo-hyroxyl ester intermediate as brown oil. CH₂Cl₂ (30 ml) was addedand the flask was flushed with N₂ atmosphere. Rodium(II) acetate dimer(8 mg) was then added and the mixture was stirred at ambient temperaturefor 1 hr. The catalyst was filtered and the filtrate was concentrated invacuo to give the crude β-keto ester as yellow oil which was used forthe next step hydrolysis without further purification. To a solution ofβ-keto ester, acetic acid (10 ml), water (5 ml) was addedpara-toluenesulfonic acid (1 mmol, 0.19 g). The solution was heated toreflux for 24 hrs. After cooling to ambient temperature, the reactionmixture was diluted with water (20 ml) and extracted with diethyl ether(30 ml×3). The combined ether was dried over anhydrous MgSO₄ andpurified by flash column chromatography (20-30% Ethyl acetate/Hexane) togive spiran ketone as yellow oil.

Spiro[5.6]dodecan-9-one (Yield: 72%)

¹H-NMR (360 MHz, CDCl₃) δ 2.47 (t, J=7.2 Hz, 2H), 2.42 (t, J=7.2 Hz,2H), 1.67-1.62 (m, 4H), 1.52-1.30 (m, 12H); ¹³C-NMR (90 MHz, CDCl₃) δ211.23, 43.95, 41.72, 38.81, 36.69, 35.39, 33.04, 26.69, 21.82, 19.31;ESI-MS: Calculated for C₁₂H₂₀O (M+H)⁺181.3. Found: 181.1.

Spiro[6.6]tridecan-3-one (Yield: 75%)

¹H-NMR (360 MHz, CDCl₃) δ 2.46 (t, J=7.2 Hz, 2H), 2.40 (t, J=7.2 Hz,2H), 1.70-1.63 (m, 2H), 1.59-1.55 (m, 2H), 1.51-1.41 (m, 14H); ¹³C-NMR(90 MHz, CDCl₃) δ 215.17, 43.96, 41.44, 39.08, 39.03, 38.76, 35.24,31.11, 22.80, 19.54; ESI-MS: Calculated for C₁₃H₂₂O (M+H)⁺195.3. Found:195.4.

The compound spiro[5.6]dodecan-9-aminium chloride was synthesizedaccording to the general reductive amination procedure. (Yield: 70%)

¹H-NMR (360 MHz, CD₃OD) δ 3.18-3.13 (m, 1H), 1.78-1.61 (m, 6H),1.47-1.37 (m, 10H), 1.36-1.29 (m, 4H); ¹³C-NMR (90 MHz, CD₃OD) δ 83.25,67.76, 67.56, 64.45, 64.25, 56.25, 55.71, 51.08, 51.06, 48.02; ESI-MS:Calculated for C₁₂H₂₃N (M+H)⁺182.3. Found: 182.4.

The compound spiro[6.6]tridecan-3-aminium chloride was synthesizedaccording to the general reductive amination procedure (Yield: 75%)

¹H-NMR (360 MHz, CD₃OD) δ 3.14-3.13 (m, 1H), 1.74-1.48 (m, 12H),1.48-1.28 (m, 6H), 1.18-1.09 (m, 4H); ¹³C-NMR (90 MHz, CD₃OD) δ 55.47,41.03, 41.01, 40.40, 39.88, 36.65, 35.82, 32.51, 28.39, 23.91, 23.87,20.08; ESI-MS: Calculated for C₁₃H₂₅N (M+H)⁺196.3. Found: 196.3.

Spiro[5.5]undecane-3-carbonitrile

To a solution of spiro[5.5]undecan-3-one (1.66 g, 10 mmol) andtosylmethylisocyanide (2.54 g, 13 mmol) in DME (50 ml) and abs. EtOH (1ml) was added KO^(t)Bu (2.69 g, 24 mmol) portionwise during 0.5 hr at−10° C. After addition, the reaction mixture was stirred at 0° C. for 1hr then 2 hr at ambient temperature. Solvent was removed in vacuo andthe resulting residue was extracted with diethyl ether (50 ml×3) and thecombined organic layer was washed with water (50 ml×3). The organiclayer was dried with anhydrous MgSO₄, concentrated under reducedpressure and purified by flash column chromatography (50%-100%CH₂Cl₂/Hexane) to give the nitrile as colorless solid (1.28 g, 72%Yield). ¹H-NMR (360 MHz, CDCl₃) δ 2.58-2.53 (m, 1H), 1.83-1.70 (m, 4H),1.63-1.58 (m, 2H), 1.40-1.26 (m, 8H), 1.28-1.20 (m, 4H); ¹³C-NMR (90MHz, CDCl₃) δ 122.79, 34.41, 31.88, 28.37, 26.88, 24.73, 21.64, 21.55;ESI-MS: Calculated for C₁₂H₁₉N (M+H)⁺178.3. Found: 178.2.

Spiro[5.5]undecan-3-ylmethanaminium chloride

Spiro[5.5]undecane-3-carbonitrile (1.77 g, 10 mmol) in anhydrous THF wascooled down to 0° C. LiAlH₄ (0.80 g, 20 mmol) was added portionwise tothe solution and the mixture was heated to reflux overnight. Aftercooling down to 0° C., the solution was quenched with H₂O (0.8 mL), 15%NaOH (0.8 mL), and H₂O (2.4 mL) sequentially. The resulting slurry wasfiltered. The filtrate was acidified with 4M HCl in 1,4-dioxane andconcentrated in vacuo. The final product was purified by flashchromatography to give spiro[5]undecan-3-ylmethanaminium chloride asyellow solid (1.70 g, 78% Yield). ¹H-NMR (360 MHz, CD₃OD) δ 3.10-3.06(m, 2H), 2.58-2.54 (m, 1H), 1.49-1.45 (m, 2H), 1.34-1.32 (m, 2H),1.68-1.64 (m, 8H), 0.98-0.81 (m, 6H); ¹³C-NMR (90 MHz, CD₃OD) δ 46.54,42.61, 37.87, 36.97, 33.25, 33.20, 28.07, 26.32, 22.83, 22.69; ESI-MS:Calculated for C₁₂H₂₃N (M+H)⁺182.3. Found: 182.3.

Spiro[5.5]undecane-3-carboxylic acid

Spiro[5.5]undecane-3-carbonitrile (1.77 g, 100 mmol) was dissolved inAcOH (20 ml), H₂SO₄ (5 ml) and H₂O (10 ml) was added and the mixture washeated to reflux overnight. The solution was cooled down to ambienttemperature and diluted with H₂O (100 ml). The aqueous layer wasextracted with diethyl ether (100 ml×3), the combined organic layer wasdried over MgSO₄ and concentrated in vacuo. The product was purified byflash column chromatography (10%-20% CH₃OH/CH₂Cl₂) to give colorlesssolid (1.53 g, 78% Yield). ¹H-NMR (360 MHz, CDCl₃) δ 2.32-2.25 (m, 1H),1.78-1.58 (m, 6H), 1.52-1.35 (m, 8H), 1.24-1.21 (m, 2H), 1.12-1.04 (m,2H); ¹³C-NMR (90 MHz, CDCl₃) δ 183.13, 43.55, 40.96, 35.78, 32.75,32.05, 27.10, 23.97, 21.86, 21.71; ESI-MS: Calculated for C₁₂H₂₀O₂(M−H)⁺195.3. Found: 195.3

Spiro[5.5]undecane-3-carboxylic acid (1.96 g, 10 mmol) in anhydrousdiethyl ether (50 ml) was cooled down to 0° C., CH₃Li (22 mmol) wasadded dropwise in 30 mins. The resulting mixture was stirred at the sametemperature for two more hours and warmed to ambient temperatureovernight. Saturated NH₄Cl aqueous solution was added and was extractedwith diethyl ether (50 ml×3). The organic layers were combined and driedover anhydrous MgSO₄. The product was purified by flash columnchromatography (20%-30% Ethyl acetate/Hexane) as yellow oil (1.40 g, 72%Yield) ¹H-NMR (360 MHz, CDCl₃) δ 2.30-2.22 (m, 1H), 2.11 (s, 3H),1.71-1.63 (m, 4H), 1.55-1.47 (m, 2H), 1.47-1.36 (m, 8H), 1.23-1.20 (m,2H), 1.87-1.01 (m, 2H); ¹³C-NMR (90 MHz, CDCl₃) δ 212.44, 52.07, 41.32,36.05, 32.39, 32.15, 28.10, 27.07, 23.60, 21.84, 21.67; ESI-MS:Calculated for C₁₃H₂₂O (M+H)⁺195.3. Found: 195.3.

1-(spiro[5.5]undecan-3-yl)ethanaminium chloride

1-(spiro[5.5]undecan-3-yl)ethanaminium chloride was synthesizedaccording to the procedure described above for reductive amination.¹H-NMR (360 MHz, CDCl₃) δ 3.14-3.07 (m, 1H), 1.70-1.64 (m, 3H),1.59-1.56 (m, 1H), 1.37-1.35 (m, 12H), 1.31-1.19 (m, 4H), 1.08-1.01 (m,2H); ¹³C-NMR (90 MHz, CDCl₃) δ 53.10, 42.14, 41.71, 36.12, 36.09, 32.16,32.03, 27.11, 24.39, 23.29, 21.93, 21.77, 16.39; ESI-MS: Calculated forC₁₃H₂₅N (M+H)⁺196.3. Found: 196.3

Example 2 Plaque Reduction Assay Using Exemplary Compounds

FIG. 1 depicts the results of a plaque reduction assay of certaincompounds according to the present disclosure on A/M2-V27A/L38F mutantinfluenza virus. The effects of the depicted compounds C¹, C², and C³ oninfluenza A virus (A/Udorn/72) V27A/L38F mutant were evaluated by plaqueformation on MDCK cells in the presence or absence of the compounds (10μN1 or dose dependent). L38F is a natural mutation in Weybridge strainvirus, which is pharmacologically silent to the drugs. Moreover, inOocyte electrophyiosiocal recording, double mutant V27A/L38F M2 channelshowed the indistinguishable channel activity and drug sensitivity asV27A single mutant M2 channel.

1. A method for treating an influenza A virus-affected disease state orinfection comprising the step of administering to a subject in needthereof a composition comprising a compound of formula (I):

wherein X and Y are independently a bond, alkylene, or amine; R¹ and R²are independently hydrogen, alkyl, hydroxyl, carbonyl, carboxyl, cyano,amino, or —CH(R⁵)(R⁶); R³ and R⁴ are independently hydrogen or—CH(R⁷)(R⁸); R⁵ and R⁶ are independently hydrogen, alkyl, hydroxyl,carbonyl, or amino; R⁷ and R⁸ are independently hydrogen, alkyl, alkoxy,hydroxyl, carbonyl, or amino; dashed lines a and b are optional bonds;and, n is 0-3, or a stereoisomer, partial stereoisomer, prodrug,pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, orN-oxide thereof.
 2. The method according to claim 1 wherein saidinfluenza A virus-affected disease state or infection comprisesinfluenza (flu).
 3. The method according to claim 1 wherein saidinfluenza A virus-affected disease state or infection comprises one ormore of pneumonia, bronchitis, sinus infection, and ear infection. 4.The method according to claim 1 said composition additionally comprisesa pharmaceutically acceptable carrier, diluent, or excipient.
 5. Themethod according to claim 1 wherein
 6. The method according to claim 1wherein said composition comprises:

wherein n is 0, 2 or 3;

or a stereoisomer, partial stereoisomer, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid hydrate, and N-oxide thereof. 7.The method according to claim 1 wherein said influenza A virus is awild-type virus.
 8. The method according to claim 1 wherein saidinfluenza A virus is a mutant.
 9. A compound of formula (I):

wherein X and Y are independently a bond, alkylene, or amine; R¹ and R²are independently hydrogen, alkyl, hydroxyl, carbonyl, carboxyl, cyano,amino, or —CH(R⁵)(R⁶); R³ and R⁴ are independently hydrogen or—CH(R⁷)(R⁸); R⁵ and R⁶ are independently hydrogen, alkyl, hydroxyl,carbonyl, or amino; R⁷ and R⁸ are independently hydrogen, alkyl, alkoxy,hydroxyl, carbonyl, or amino; dashed lines a and b are optional bonds;and, n is 0-3, or a stereoisomer, partial stereoisomer, prodrug,pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, orN-oxide thereof.
 10. The compound according to claim 9 wherein X and Yare each independently methylene or ethylene.
 11. The compound accordingto claim 10 wherein X is methylene and Y is ethylene.
 12. The compoundaccording to claim 10 wherein at least one of R¹ and R² is hydrogen. 13.The compound according to claim 12 wherein one of R¹ and R² is carbonyl,amino, carboxyl, cyano, or —CH(R⁵)(R⁶).
 14. The compound according toclaim 13 wherein n is 1 or 2, except that if n is 1, both X and Y aremethylene, and both R³ and R⁴ are hydrogen, then one of R¹ and R² is notcarbonyl.
 15. The compound according to claim 13 wherein one of R¹ andR² is —CH(R⁵)(R⁶).
 16. The compound according to claim 15 wherein one ofR⁵ and R⁶ is amino.
 17. The compound according to claim 9 wherein saidcompound is:

wherein n is 0, 2 or 3;


18. A composition comprising a compound according to claim 9 and apharmaceutically acceptable carrier, diluent, or excipient.